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Full text of "Study of the dissociation of some strains of Erysipelothrix rhusiopathiae of swine"


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fl» Bffi»«t of MftrtMit Broth (pn 7«6} In 10 oe« 
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COKCLtJSIONS ..••• • •••••• 

IXSSOUlSWSL CITED •*••»«•♦#••»••»•••«•••••••*•••••••♦ 





After irlcvngiht (1^21) and m "Smlf {19®t| published 
the results of their ©xperlmsnts that rough anA ««ooth colony 
toxmm nay he obtained froia the wan pure culture of bacteria* 
there has been an ineroaslns attention and interest In the 
studies on bacterial variations. The studies deollag wtUk 
tills subject desigaats ©ertaln more or le«i ordered aodifl- 
eetlons In beeterial cultures • c^hangea ifeleh In meaiy cases 
»ay be x^redlcted and controlled and ahlch are Intimately 
MMeeiatea irl^ eell iao(rp!iology» colony form, virulence , 
blocheodeaX babsTlor mi& aaans&eele reactlona« 

Wyrtrj (1987) publififoed a complete and excellent review 
dealing vlth the various pmm6B of the pheaoaaaa of mlcroble 
Aiaaoelatlon. A ot^plete review of literature Is not gtiwa 
in this paper. Forty-six r^orences dealii^ «lt3i the sub- 
ject «ere read* Froa the lltei^ture it Is condviaS tlwt 
all aiMMdes of baeterla aay tm dissociated into variants, 
and that this dissociation aar %» tmM» reversible by sub- 
jecting the variant to different enviroffinaatel conditlc»Mi« 

Xnvestigatars differ widely in their opinions as to ttie 
•Sgnifi canoe attached to variation of tectoria. 9mm In- 
vestlgatera lllce De KTulf , S<aratze and others believe that 
^ bacterial variation la a ^fisaracteristic which is inherent 





witailn all bacterial cultures and tJjat this (Siaracteristic 
la transferred to subsequent generations. Others regarded 
Yarlation as due to en-ylronmental conditions to which the 
flTiKiiim is subjected* 

The ccmception of bacterial variation has upset ^m 
wjucaaorphic theory of Cohn and Kooh, as reviewed by Jordss 
«Rd Falk (1988), 1*10 regarded Gutter! a to be ehametsrized 
trtth rather rigid fixity to their specific type as pertains 
both to morphology and physiology/, any deviation fran ths 
a^naol vsre regarded as oontaminations. 

The appearance of a variant is regarded as a fact but 
there has not been a satisfactory explanation ^y a smooth 
variant becones rough and vice versa* East-^ood (1932) Imm 
at^orpted to explain the fact on the basis of certain pecu- 
liar characteristics of grot^th. He stated that there are 
nmJKHis to believe that growth depends cxi a r3rthmic cycle of 
synthesis, peculiar to living, protein, hereby at each sio- 
•^ive stage one particular- "building stone* is selected 
for synthesis with rejection of all others. "Hie enzymes are 
oonsidered as centers of "chenloo-phyelcal" activity and the 
eaergense of variants may b© explained by the liability of 
these "centers'* to cdiange under "elMndLco-physioal" Influ- 
esioes* 

Studies on the dissociation of Swine erysipelas oi^an- 

have not been fiotmd recorded In any of the literature* 





', Gaiger and DaTles (1932) stated that rough (R) and 
>th (S) Tariants occur on aear laedia, and Kinder and 
Bttkins (1931) claiiaed that they isolated a vimlent strain 
of E, rhus^^ mHu^ f roia an in^fttlMi «Bong f isheroen hand- 
ling live fish and after passing the organism on colture 
media for seven weeks, its virulence for ialo« vets decreased. 
In 1927, rJayson working on the Bacillus of aenwe septicenia 
produced colonies appearing like bone lacunae after ttali 
eslatine stab ealtures had been held at approxlaately 20«C« 
for froa four to six weeks. 

Because of the wide distribution of S^. 



In nature, its economic importance, and because De Kmif in 
1921 found that rabbits inoculated with his virulent G, 
cultures of B. leT?iseptieuia resisted lethal doses of his 
virulent D. cultures, led to the investigation* 

QQWif^BS OF TISS STRAINS VSW 

StiBlns 3506, 2310 and pH of E. rhusiopathiae vmre ob- 
tained by Dr. H. T. LienhaPdt, Head of the Department of 
Patiiology at EMiMts State College from the Pitman-ISoor© 
Company, Indianopolis, Indiana. These strains ^rere isolated 
froa eases of Swine erysipelas in South Dakota. 

Strains 106, 14159, and 13843 of E. rhusiopathia« 
isolated from hogs died of E. rhusiopa'Hiiae infection 





brought in the DepartsMiit of Pathology at Kan«a« State Col- 
fmet jfaations totms in the State of Kansas* 
strains 1146 and 1147 of 3alaonella suit>estifer organ- 



wero obtained frraa Dr. C, A, Brandly of tlw 
of Bacteriology at Xanaa* State Colleg«#. 

Strains 7, 8, and 17 of Pasteurella organisufli were 6b- 
tftiaea from Dr. J. P. Scott of the DepBrtaent of Pathology 
•t Kansas State College • These strains were used in a 
study of shipping ferer of cattle in the State of Kansas* 



ftroughout this imrestigation, liquid media were used 
because there seeiss to be a gMMural agJNNMMttt anMlg investi- 
^Uaem i&o have be«i vorlctag oB tlil» pHww w an on that in such 
an environment, changes may be broiight more readily tlmn on 
solid nedia. This fact was bixnight out by Hadloy (1927) la 
his reTiew on nicroblc dissociation, Th« obserrations of 
Arkvright and others also eaqphasized tSm greater tendency 
for variation during gtovlli In liquid iaedia» 

Six strains of Smtne erysipelas, two strains of Sal- 
monella sulTJestifer . and three strains of Pasteurella organ- 
inui were used* The Salmonella guipestifer and the Pasteur- 
ella oi^aniBOB weve used as a cho<& or control. Previous 
to their use in this study, they were plated on modified 
salts agar because "ttie Swine erysipelas and Pasteurella 





8 



tsns seened to grow better on this medliEn than on 

iratrlent ttgar. This is a aodlficntion of ^to mineral salts 

•esr a»4iiBii described by Scott (1930). Its oogq^ocitlon is 

as follows: 

PiBpton© 20.0 croms 

Sodlun chloride •••••••••«•««•••••••**• 3.0 grmae 

Potassium bicarbonate ••••••.•••••••••• 0.5 grans 

Potassium el trate •.......•••«*•»••••«• S«0 grans 

IWionlicn hydrogen '^ •••• •• 0,6 grans 

Agar (Dlfeo purified) •••••••••.. SO.O ^mm 

Glucose ...«.••.••••• 0,5 gSWW 

Glycerol ••.••••..•.•••••••«••••••••.•« 5.0 cc» 

Distilled witer •«« 1000.0 cc, 

above in^redioits were placed in a 2000 cc. flaslc and 
in an Arnold stcrili^ser for one hour. Without ad- 
^-osting its pr, the mixture too placed In 100 cc. bottles 
and autoclaved at 15 pOfflsAs presstir© for on© hour. T/ell 
isolated tj^ical smooth colonies sera selected and inooulat- 
tA in nutrient broth. The broth cultures wore incubated at 
57* C. for 24 hotn-s and the abore process repeated. This 
jiToqaf of replating and reisolating was repeated f iTe dif- 
ferent times. In order to doubly insure the purity of eMil 
strain, fermentation tubes containing glucose, lactose, 
aaltose, sucrose, dulclte, xylose, arabinose, raffinose, 
amose, dextrin and inositol were inoculated and incubated 
at 37* C. The reaction was read at 24, 48, and 72 hour inter- 
"vals. All test media were inoculated alth 0.2 cc. of a 24 
hour culture of each strain of organisms used in this in- 





vestigatlon. 

At appropriate interrols aodlfied salts agar plates 

■iMHdDsA mtA sssalned for t3rp88 of colonies after 24 
48 hours Incubation at 57' C. This procedure pGmltted 
one to follov the ttme^e restating from cells near tiss yaak 
of developnent as well as at later periods. The colonies 
IMMNi MMdtesft vrlth the aid of a low power binocular niero- 
scope. This mxa found to be useful especially in the cosa- 
pariaon of colonies, 

De Kruif (1921), Dulanoy (1988), Soule (1928) and 
others tevs tfMm that guinea pigs, miee, and rabbits in-> 
oculated xsith tbe rough foma of t!is <wrgii|wim «lth vhieih 
they vsrs working, x^^teeted Xtiose animals sealnst ths 
lethal dose of the smooth fanas* Therefore inteirest xms 
stinnilated In determHiing the variation tendency of Swine 
•ryslpslas oi^asifflas* sAd also an effort was zsade to pro- 
duce an avirulent form T^iich tjould give protection egaittSt 
fto Tlrulent tsrpe. 

Six strains of Swine erysipelas organlSBS wan streaked 
on modified salts a(^flp plates and the types of colonies 
found were studied, estimated and recorded as a per cent of 
all colonies present. 





nBCEi?Tioi7 OF ccumm 

ftaPoughout thie Investigation, it was ofeserred that 
13ie chan^ froB wmoo'&k to rough is gradual, contimKms, andl 
ireversible. It is not a direct change and does not occur in 
— mm l distinct abrupt steps. The tenaa Steooth (S), Intep- 
iBMftlftte (Z), and Rough (B) are used to desigmts tim elhiet 
tfptm of colonies found. This arbitrary separation into 
lAt^Sories does not reiLlly Make clear the true course of 
if tie* ColoBles appear ^ieh are very difficult to plaot 
In either the intermediate or rough forms. One Inyeetigator 
aay prol^ably place th&n as interr»dlate famm irtille another 
■ay desigmt* tlmi am rot^h typ^s^ so that the results on 
dissociation are coagp^mble cmly 1b a e«ne2^1 way. 

Smooth (S) form of g. Mrosiopathiae 

fl» Oftooth colonies of Swine erysipelas organtaB ar« 
aaall, round and convex homogeneous disks. Ibe laargins are 
regular In outline. The surface is saaooth containing few 
AliA mrtf seen under l^e low power of the aicro« 

u They grow diffusedly in broth and have a unifom 
tcrbidity in 0.85 per cent NaCl. 

Htm indlTidual cells of the smooth colony are Gran 
I)OsitiTe slender rods and are arranged singly. They do not 





11 

have spores or capsules. They do aot ferrsent any of th» 
mamstB Msed in this inrostigation. Th^ do not produce 
indol and do not Iwaoly;^ blood. 

Ro^gh (R) Form of E. Rhtisiopathiae 

The 2roiigh (B) colonies of Swine erysipelas organisa 
are flat, coarsely granular and larger than tlie «K>oth (S) 
colonies. The raargins ere very irrornaar and broken in 
outline, Flocculeut growth appears at the bottom and side 
of tl» broth tuto, the upper portion being clear as if it 
had not been inoculated. Spontsuieous clumping oecura in 
physiological salt solution and it is impossible to obtain 
a uniform suspension of the organiams. This is due to the 
concentration of salt in the solution in the opinion of 
AstaTriglit* 

Iha individual cells of the rough {R) form ara Grtn 
positive, slender rods, longer and slightly larger than the 
individual cells of the aaiooth (S) form. They oceur in 
Miises and in long or short chains. They dc not hav« 
apores or capsxilea. They do not ferment any of the su^^ars 
used, do not produce indol, and show no hemolysis in blood 





Inteisodiate (I) Fom of £. Rhuslopathlae 

iMiy colonies of difforeat appearance are included 
the intermediate U) type. The colonics have «*d@s» 
or sectors. Frequently the sector appears to consist of 
Tery irregular line* or to be coars«iy granular. They are 
iMKfB'ft tUMi tlMi SBOOth (S) type. In laany cases the inter- 
mediate (I) colonies are hard to distinguish frosn the rough 
(R) colonies. They more frequently revert to the smooth 
(S) fo« ilien planted in broth* 

The individual cells of the intes»ediate colony form 
are dresa. positive, slender rods ocesslonally occurring In 
chains or singly. 

Colonies of the interraediate type frequently occurred 
as tl» first notification of a change and developed before 
the rough forcis occ\irred. 

wcpmmmsAL methods used to incite s to a ©lasociATioH 

1b« ittamt of igiug anooth BBi«terlal Culture 
iM liutrient Bro^ 



The nutrient broth t^s pwt§tsm^ Mi follo\r7s: 10 
peptone, 3 grams beef extract, and 5 grains sodium chloride 
were placed In a two liter flask and 1000 cc, distilled 





v&ter «88 added. The mlxtior© vas placed In en Arnold 
sterilizer and steoaed until the ingredients ware thorcmghly 
61»8olTed. fhe loss of -rolDMe due to evaporation tms re- 
plae«d by the addition of distilled \7ater. The reaction of 
the solution lias adjusted to pTI 7,2 by the colorlraetric 
aethod. The tooth vate placed in 125 cc, Srlenneyer flasla 
in 100 cc, aHOunts and autoclaved at 15 pounds XTessiire for 
*iB MmtaMft« flMl reaction was not readjusted after steril« 
Ization. 

A number of these fluids were inoculated with six 
strains of E, rhusiouatliiae . t«o «K>oth strains of 
[>la suioestifer and three smooth strains of Pasteur- 
«lle organiODis. The broth cultures were incubated at 37* C« 
during the r«malnder of the experiaent. At a 15 day inter- 
val, during this period, a loopful of the material froia 
each strain was streal«d on modified salts agar plates. 

The results of tola eaperlment ar© recorded on Table 1, 
VM) effect of aging E. rhusiopitthiae In broth varied in th» 
degree of dissociation of the different strains of the organ- 
San as shown on the table. Strains 2310, 3506, 13843, and 
pH shoired dissociation at the end of thirty days, i^lle 
strains 106 and 14159 did not show rough colonies until the 
fartywfifth day. The table also showa that the Serine 
erysipelas organisms were dissociated in lese tiiae than the 






ttStHe 1. Percontag© cf the R form of F.. f^mmpt.hiaft 

l»y the ©ffeot of asins in nutlfdnt 
teoth adjiisted to pK 7.2. 



BiBPiod cf incubation (days) 


19 


30 


45 


60 


120 


Strain 




Percentage c 


f R for 


aed 


106 S. rhnsioTjathlao 


lOOS 


101 


15R 


eos 


lOOS 


2310 ^, rhtislopathia© 


IOCS 


5R 


25R 


WR 


lei 


5506 '£, rhusio-pathiae 


lOOS 


IR 


30R 


75R 


lOOS 


13845 S, rhusioDcthiao 


lOOS 


m 


40R 


lOOR 


5R 


14159 111. rhuB lopathiae 


loos 


4M 


20R 


80R 


1003 


pH t. rfeusioi>athlao 
1146 B. salve strr<-T .■ 


lOOS 


ZR 


50R 


lOOR 


7S 


lOOS 


lOOS 


3R 


25R 


lOOS 


1147 ^. Btiipe£^-"'or. . 
7 rc.steui-clla 


loos loos 

lOOS SI 


lOR 

IE 


50R 

30R 


lOOS 
lOOS 


8 P«»tet2rella 


100& 


41 


7R 


50R 


lOOS 


Vf Peisteurella 


loos loos 


51 


20R 


lOOS 





lella stiipestlfer and Pasteupolla organlsrss, Th* 
-per e©nt of R fonae wore obtained on the sixtieth 
day. After this period it seeras as if the B fonui wtnem 
gradually reyerted to the S form. 

The above ejperiaent confirmed the obaitiittions of 
menE^lfat end Pitt (1929) working tTith tlia typhoid and para- 
tyirfioid orsanisms, and earlier observations by Jjtosright 

(1921) that B forme can usually be found in old broth cul- 
tures orlglQally inoculated with the S form. The tine re- 
quired to produce the chuBge iBried fron 2 or 3 wmUcm to 2 
to S awnths depending upon the organism and t3ie strain used, 

loKking with the Bacillus of zmMlit amptlmmiM M Kruif 

(1922) produced dissociation by a^ing the organism in plain 
broth. Goyle (1926) obtained rough variants of B, typhosug 
end B» enterditls Gartner from old broth cultures by plat- 
ing on agar and selecting colonies. Aging broth culture 
wtm fxao of the factors in inciting dissociatioa Bwaitloned by 
Dulaney while working with B. coll oooEiunis in 19^, and Li 
in 1929 produced rough variants of Hog Cholera bacilliw 
twem h&Bt Infusion broth inoculated for three weeks* 

Dissociation is usually most narked when conditions 
are unftivorable to the growtJi of the orgmint* 





The Effect of 10 Per Cent HoEiologous IcEaune Serum Broth 

In Inciting Dlssociatioa 



IPhB <«rir* macws. iised in this inrestigation «&« (^taln- 
«ft from rabbits inoculated trtth foxraalized Swine erysipelas 
flVanlsms standardized to tube 10 of ?!cFarland»s nephelo- 
wmt«t at Intervals of one voelc* The rabbits w^m i^vsa fivs 
inoculations subcutanooiisly doubling the dose at each «io- 
esBsive inoculation. %» days after the last injection the 
rabbits were bled frcsi the heart. Agglutination tests iTcre 
run to detoTmine the titer of the sera. To ^eh tube con- 
taining 9 cc. of nutrient broth was add©« 1 e«. of h<»aologous 
le senna to (^re it a concentration of 10 per cent by 
The tubes of serum brotlJ swre incubated for 24 
at 37*C. to test for sterility. Kisy were then in- 
oculated site the OTBOoth strains of the Swine erysipelas 
Oiipttfsws iised in this inrestl-ratlon. The cultures sere 
incubated at 37" C. and at sKpspopriat* tBtorrale, they were 
mlamBk od. on modified mXtm agar plates to detenaine the 
typ&s of colon iss« 

Tte results of this experinent sere in accord irttb ttie 
observations of other investigators. Stryker (1916) ob- 
tained a rough strain of pneunococcl shich was quite stabla 



MM 





I^y groviBg the or&atiBa. in homologous iMmine serun, Grif- 
fith (1927) fm» the first one to Intentionally WM MitiBertW 
pxvipered by -ei© lanainlzation with hooologoiis antigens for 
#t««ociatine the pnetHaococcus orsanisn into its related R 
tnMW« Later similar results nere obtained by Arkwplght and 
Pitt (1929) working with E, typhi , by Dulaney (1926) work- 
ing with E. coll . and by Soule (1928), Li (1929) and others 
iftD worked with paratyphoid organisms, Soule (1928) also 
fliiteined the same results with B. subtil is > 

It Wtts fotmd that 10 per cent hcsaologous iiassone semn 
broth is en effective medium In inciting S to R dissociation 
Ott S»ine erysipelas. Salmonella suites tifer. and Pasteurella 

lisns. 

The Effect of Nutrient Broth (pH 7»6) in 10 cc. and 
100 cw. Amounts cai the Dissociation of 

E« Khuslopataiiae 



nutrient broth vised. In this expertoent was prepare* 
similar to that mmH. in previous experla»nt. The liquid 
M»Alm «w edjtisted to pH 7.6, plaeoA ta tubes and in flatriks 
in 10 ed* ttiA 100 ec. amounts respectively and tatoclaved 
at 15 pounds pressure for 45 minutes • 

4 BMiber of the tubes ond flasks oootainlns the neditai 
w»re seeded vrith six smooth strains of Swine erysipelas. 



17 





n 



©aootli strains of Salnonella stilT>estlfer . and three smooth 
strains of Pasteurella organisms. The cultures were incu- 
bated at 57* C. for twenty-four hours, after which tl»y wqt9 
placed at rooai t^ajperature , At appropriate intervals the 
cultures were rt ai cgn $BudL a loop from each tube and flask 
were itrnjlnnrl on w>dif led salts a^:;ar plates to determine the 
yariotis foxsis of colonies, 

AS shown on table 2, no I or R t3rpes ^sre produced di2P- 
Ing th« first ten days of itaB asiparinent. At later inter- 
Yals I or R fonis were observed in the tubes and flaslM 
eultures. A gr^iter percentage of R type* w«re obtained in 
the flask cultures than in the tube cultures. The greatest 
number of rarlants were produced at the end of sixty dajrs 
«tt rooo tejaperaturo, AK)roxifflately 35 per cent R colonies 
were obtained fron the cultures in 10 cc. anoimts while in 
the larger volurae of laedia, an owerage of 60 to 70 per cent 
E colonies were produced. These obeerrations were in ac- 
cord with the findings of Soule (1928) who has sho^m the 
•ffect of a large volucic of liquid flSAfa upon the dissocia- 
tion of B, sub til is. and Hadley {19S7) who stated that the 
decree of dissociation after a given tine is greater in the 
volume than in siaall voluEie of aedium. This is 
it to be due to the fact that the period of growth has 
been lengthened. 



18 



■ 


• 






• 




• 


1 


• 














m 

19 




IWble 2. 


A coaparison 


of 


the effect 


of awttrient 


broth 




(pH 7.6) in 10 oc. 


and 


100 cc. aawMVtS to incite 






dissocif^tion 


of 


E. rhusioT>at*ltte< 


u 






Period of 


incubation (days) 


10 50 


50 


70 


140 


Strain Aaount of broth 




Percentas© of ^ 


', I, 


and R 


106 


10 




lOOS SI 


lOR 


30R 


lOOS 






100 




loos 2R 


80P 


50R 


lOOS 




Kao 


10 




lOOS 51 


15R 


40R 


1003 






100 




1003 4S 


30R 


85R 


31 




5506 


10 




lOOS 101 


7H 


50R 


1003 






100 




loos IH 


55R 


80R 


1003 




&88Ci 


10 




lOOS 2R 


IbK 


60R 


71 






100 




lOOS 7H 


40R 


95R 


» 




14159 


10 




1003 SI 


5R 


35R 


lOOS 






100 




1003 SB 


25R 


75R 


1003 




10 


10 




lOOS SR 


2^ 


60R 


lOOS 






100 




lOOS bK 


50R 


90R 


2R 




1146 


10 




1003 lOOG 


IR 


lOR 


1003 






im 




lOOS 1003 


lOR 


25R 


1003 




1147 


10 




lOOS 1003 


SR 


30R 


lOOS 






100 




loos lOOS 


lOR 


45R 


1008 




f 


10 




lOOS lOOS 


11 


20R 


10«8 






100 




1003 31 


SR 


85R 


lOOB 




8 


10 




1003 lOOS 


9H 


25R 


lOOS 






100 




1003 41 


lOB 


(OS 


1008 




17 


10 




1003 lOOn 


71 


lOB 


loes 






100 




1003 100.. 


6R 


25R 


1003 


• 

















r 





It wmB dbaerrod. that tlte dissociation Ifi the E, 
x^usioTHStthlae as tmll as in the SiftjHDnella and 
organ IflDUi i^ppeared to be s to I to R* After the arganlao* 
wire in th« dissociation nedia for a long period of tiao 
tl» rerei^lon appeared to be R to I to S. Hadley (1927) be- 
lieve* that tbmTB Is probably always an inters^diate fom 
apparently both the progeny of the S and the prosenitor of 
^le R« He sogBBAta t^at &• B type ie not a direct but an 
indirect product of S and Its production cooea about through 
the functioning of an intenacdiate stage of culture develop- 



Effect of Too Concentrations of Peptone and 
Variation of Temperature tJpon tte fltaooth 
Strains of S, Rhusiopathlae 

One per cent and five per cent Bacto peptone solutions 
prepared and adjusted to pH 7*6 by the colorlmetrle 
aat2iod« The i^dia were tubed in 10 cc» amounts and auto- 
leafed for 30 minutes at 15 pounds pressure. 

A. wamSber of tubes of each concentration of ^tm peptone 
solution were Inoculated elth six saooth strains of E« 
rhusiopathlae . tee eraooth strains of ^latonella sulpeetifer . 
SBd throe snooth strains of Peeteurella organisns. One set 
of eultujws eere incubated at 37** C« and another set wbm in- 



to 





>d at SO^C. At an interval of 15 and 30 ^^7* the eul- 
ive thorotiglily iftixed by shaking and a loopful trcn 

eolture wete «^r9el9»d on modified salts a^ar plates an<l 
>d for various t3rpes of colonies. 

results of this experiment are tabulated on tables 
S and 4, A* tfiom on *1» tables, more R variants wsre pro- 
duced in cultures incubated at 37° C. than those at SO'C* 
indicating that hl^er temporattti* offers a greater atiiaalus 
for di«Bociation» 

A grwtter decpre© of dissociation trecurred in 5 per cent 
Bacto-peptone than in 1 por cent peptone. Dissociation in 
5 per cent Bacto-peptaae tras variable depesftms ^o^eat tbm 
liTginffniii and strain used. In 1928, Soule isjorking with B, 
gubtilis observed that no dissociation occurred at 5*C., 
vHile variations occurred at a temperature of 25* C. He 
found that gg^ylTmim dissociation was obtained at a tempera- 
tnre of 45*C. !>alaney (1928) stated that temperature and 
moisture {4M to 45* C.) stirmlatBd dissociation of B. eoli . 
Nungester (1929) while working with B. anthracis found ttiat 
• iMZk«d decrease in dissociation activity occurred at 26* C. 
and 37*C,, while at 42* C. he observed the greatest decree 
of dissociation activity. 

Koser «id Styron in 1930, lAlle woilcing with B. dysen- 
teriae stated that the organisai did not show any tendency 
to dissoelatitm in 1 per cent peptone irrespective of pH, 



p 


• 


• • 


• 


^ 


• 








22 




TMHe S. A eocrpctrlson of tlie matbrnr of R foms of 




!• r^isiopathiae pirofiaewfi fer the cultivation 






or vm S type in ttjo concentpations 


of 






peptone adjusted to pE 


7.6. 








Strain Period of Incubatlor 


I at 37' C 


15 days 


30 (inys 60 days 




Organ isms grown in 


1 ^ neptonfi 


106 IQOQ 


lOOS 




IQl 




2310 1000 


11 




m 




3506 IflOt 


lOOS 




71 




13643 WOB 


looc 




151 




14159 SOOS 


lOOG 




41 




pH loos 


51 




IB 




1146 IMS 

1147 ms 


lOOS 




lOOS 




lOOS 




SR 




7 loos 


lOOS 




81 




• loos 


IMS 




IR 




If lOOS 


I0OS 




lOOS 




Orga&iaui grotm in 


5 ^ peptone 


106 lOOC 


21 




8^ 




2310 lOOC 


101 




aoR 




3506 lOOS 


SR 




40K 




15843 lOOS 


m 




60R 




141§8 lOOS 


in 




9SR 




im loos 


3R 




mm 




1146 SiOOS 


lOOS 




15R 




1147 lOOS 


151 




SOB 




7 lOOS 


lOOS 




15R 




e um 


lOOS 




25H 




It 1606 


lOOS 




lOR 


• 










. 


' 












1 



ss 



iWlde 4* A conparison of the ntiober of R foras of 
^ '"^TBttW^^^'^^ produced by the cultivation of 
twl W iype in two concentrations of 
I)eptone adjusted to pH 7»6, 



StraiB 


Period of incubation at 50** C. 




15 days 


30 days 


60 days 




Organ isms 


grovm in 


1^. peptone 


106 


aAos 


lOOS 


IOOS 


2310 


t908 


1003 


81 


3506 


lOOS 


lOOS 


51 


1380 


2008 


10OS 


21 


14Iii 


190S 


loos 


IOOS 


^ 


XOOS 


IOCS 


IB 


1146 


1003 


iSS 


1003 


1147 


100 s 


1003 


7 


lOOG 


lOOS 


101 





lOOS 


lOOS 


IR 


If 


XOOS 


lOOS 


IOOS 




0rgani«a8 


grown in 


59^ peptone 


106 


lOOS 


lOOS 


5R 


2310 


loos 


1003 


7R 


3506 


lOOS 

SSa 


1003 


lOR 


13843 


ioos 


8R 


14159 


XOOS 


Sos 


» 


ia£ 


lOOS 


18B 


1003 


lOOG 


IB 


1147 


lOOS 


IOCS 


4R 


f 


1003 


IOOS 


iB 


• 


lOOS 


IOOS 


SB 


19 


lOOS 


IOOS 


IR 






fhty found that those gross in 5 per cent peptone foxnnt 
intermediate and rotish colonies. 

De Kmif (I9S2) 4JtoMiw4l «hst incrMMliig concentrotioiHi 
9f peptone accelerated the app©«nBice of the granular form 
of B. leDiseptleun. He stated that a 20 per cent solution 
edf peptone was nearly as effective as 5 and 10 per cent 
solution. 

There was a greater Tariatlon in tjrpeB of colony and 
Srottter production of H "roriants for Sirtne erysipelas, 
StiyHMtik^ suipestifer. and Pasteurella orgemisras in 5 per 
^nt peptone than in 1 per cent peptone solution. Dissocla* 
tioK in 5 per eent peptcHie warn Tariable dep^iding v^khi t^ 
and strain used* 



The Effect of Beet Infusion Broth Adjusted to Taryli^ 
Hydrogen ion Concentration <m the S Torm 
of B> Bhusiopathlae 

flm preparation of the beef infusion broth is as fol- 
loi»: Place 1000 cc. meat infusion broth in a ^00 cc, 
fStttft and add 1 per cent peptone and 0,5 per cent sodium 
chloride. Heat the mizture until the inc;redi^it« «m 
thoroughly dissolved. The solution was filtered through 
filter paper adjusted to pH 6.6, pH 7.0, pE 7.6, and jtfT 6.8, 
tubed in 10 c©« amounts and autoolaved at 15 pounds pressure 
for ^irty minutes. 



24 






25 

A wmiSteT of the tubes of eultiire aedia of varying hy- 
arc^en Ion concentration were inoculated with six anotft 
•lerains of Sirine erysipelas » two smooth strains of S^aaonol|K 
WfAimmtlt<Mr aud ^etme smooth strains of Pasteirrslla or^an- 
isos* Tim cttltttres w&re luetftjated at 37*C. At affftjprlate 
intervals a loop from mailti culture ms stra«lB»4 on modified 
saltB asar for the deteminotloii of ▼arious type colony, 

SSbe results of this es^riiaant are showa on table 5. 
9tlt»e erysipelas ana Basteurella orgwajlip failed to 

in the aoid imdirm mn» Xbs Sg|MHlh3^ sui-seii^te^ 

«^X* Ho R variants appeared during Vim tU9% tamty 
days while at the end of sixty days^ R types ^sere obtained, 
fhe culture laedlum adjusted to pll 7,0, pJI 7.6, and pK 7.8 
i^eared to be very favorable for S to R dissociation of the 
matm tsryelpelas, Saliaonella JBttttUEK* ^^ Pasteurella 
OT'ganiasis* 

The results of tiiis experiaittt «bi« »imilar to the 
findings of several investigators, Soule (1928) working 
*1*^ B. subtills o%«©rv&d that an initial reaction of a 
medium of about neutrality Is laost favorable for S to R dis- 
sociation. In 1928, Dutton stated that an allcalin© nutrient 
iplHl |§ <B essential factor in furnish log the proper en- 
viroifflent for the dt«»«iatioa of stroptocccci. Hitfley and 
(1951) dissociated the S form of B. typliosus in be«f 



p 

• 


• 








• 


U 




Table 


5* A Mtaparlaon 


of the 


nvaxbwt of B Fonns 


of 




E. 


rhiisioT>athiae obtained by the cultivation of 




the 


S f orra in beef infusion 


broth adjusted to 








TBpyiag hjrdroeen 


ion concentrations. 






Strain 


Days at 37*C. 


Initial pE of 


beef infusion broth 


6.6 


7.0 


7.6 


7.8 


106 


XO 





lOOS 


IOCS 


lOOS 






fiO 





lOOS 


IOCS 


lOOS 






60 





30R 


35R 


35R 




2310 


10 





lOOS 


lOOS 


lOOS 






20 





lOOS 


1008 


lOOS 






60 





50R 


45R 


42B 




5506 


10 





lOOS 


lOOS 


lOOS 






20 





lOOS 


lOOS 


1006 






60 





45R 


4sm 


48!t 




19M9 


10 





lOOS 


1008 


lOOS 






20 





lOOS 


lOOS 


1003 






60 





55R 


60R 


50R 




14169 


10 





lOOS 


IOCS 


lOOS 






20 





lOOS 


loas 


lOOS 






60 





40B 


SSR 


4m 




ps 


%Q 





lOOS 


lOOS 


'Wdm 






20 





lOOS 


lOOS 


lOOS 






60 





35R 


4m 


30R 




1146 


10 


lOOS 


lOOS 


1008 


lOOS 






20 


loos 


lOOS 


lOOS 


lOOS 






60 


loos 


20R 


leu 


lOR 




1147 


10 


loos 


lOOS 


1008 


lOOS 






20 


lOOS 


lOOS 


lOOS 


lOOS 






60 


lOOS 


25R 


30R 


sn 




7 


10 





1003 


lOOS 


1008 






20 





lOOS 


lOOS 


1003 






50 





20B 


808 


Ifll 




8 


10 





lOOS 


1008 


loos 






SO 





lOOS 


lOOS 


lOOS 






60 





30R 


mm 


20R 




It 


10 





lOOS 


1003 


lOOS 






20 





lOOS 


lOOS 


lOOS 






60 





15R 


25R 


20R 


• 
























^ 






m 



infusion broth adjasted to pH 7.8. A nwatral or slishtlj 
alkaline •ne'^tone solution was used by Koser and Styron in 
1930 for aisscciating B> dysenteriae . 

tBS JSFLWSSCE OF WCTSRIOHUGl OH B4CTEBIAL DISSOCIAJICW 

tbar* Mm ke«B avweral theories ad-fttiM»e4 as to the 
nature of bacterioB*««» and the aMMoUai of ttw lytic ac- 
tion, t»^relle (19S6) regardied bacteriophage to ^ aa 
oltra microscopic f iltrable virua which acts aa a pftrasita 
OB bacteria and mamum their destruction by lysis* It 
■sltiplies only in the presence of baeteris. He stated that 
bacteriophage is the eimse of bacterial Mtttations. 

Bordet and Ciuea (19S8} believed that bacteriiq^MfS is 
an autolysln arising in the bacteria theaselrea. This 
theory is moat accepted by aajority of investigators, 
Zixtsssr (1987) TSgttfted the lytic action ae an enzyme prob- 
ably active upon the lipoidal eonstifewmts of tfee bacterial 
limiting ssaslRnsies and structures that hold them together. 
Re believed that this enzyme Is liberated by all bacteria. 
Hadley (1986) statea tti«t bacteriophage is a prodnct of 
bacterial dissociation. There is probably a relationship 
shich exists beteeeai filtrabllity, dissociation and b«cter- 
(M po^tea out by aoAiiSfjf and others in 19S1 irtillsi 
slth their foita of Shiga bacillus. 





Several inrostlaitops reportod that they have succeed- 
ed in producing bacterial dissociation tiritli tSMi aid of 
bacterlophase. In this investiGation the presence of Imic- 
terlophage could not !» deraonstrated by any of ttie tests 
wmSl If- Bodley (1927), liadley and Jiaenez (19S3L} «atd Plas* 
tridge and Rettger (1932)* It moB fonnd tliat dissociation 
at tecterial culttorec asy t» pirodMs«ft vlthout ti»f inflttenee 
•f iNicterlophage. 

B TO s mnasim 

At oii« tlBie several imwBtStsi^ttxtB like Selmtae (19£L), 
a* EMlf (1922), and others believed that tlie roogh (R) 
ttanm of bacteria <K>iild not b« miOm to revert to the MDOttt 
(S) typmu Beoent works have ^o«q that the B to s rever- 
sion aay be aceosKillshed at taiie inreseait tlas by a "warlety 
fxf asthods. 

In Artc«ri|^t*s vork in 192I» R taram of the t3rphold 
bacterivaa and of ths psratyiaioid B tMStwrf w&re mde to 
ytlrlA mn almost uniform tmrbldll^ by dally saboultnre ia 
broth, thus indicating the loss of the E cdiaracteristics and 
reversion tB W Ut'J s the 3 fona. Griffith (1927) caused a re- 
vaopsion of rou^ piSBBococci to the smooth fona through 
P ffiSSSgW in homologous tannine serum. Orcutt (1929) found 
that variants of BaetSKfi i al—ti f er that produced granu-^ 





fi *o S reversion in pnemo«(MMl by «QiXoylxig Griffith's 
aiAtboa, and throtigh anteal passage. Bisllaflim and Oallo 
Jwported that R forms of Braoella organlaB wme ehangad te 
S type ^ T/tLwrnm l^isoagh gaisea pigs. 

The aethod used in this lEvestlgatlon In attempting to 
produce S forrrs frco R WPlemte, was a successioaa of dally 
transplant* of the H types in beof infusion broth, saal 
solid salts agar and serum broth. Six stabilized rou^h 
iWKPlants of S. rhusiopathlae vere selected ar^ used P vme^ 
this InTestigatlon. The results were recorded tm S, I,^ 
R accordlns to the types of eolonies observed on siodi<» 
fled salts a^ir plates. 

B» Sffeet of SueoMsi've I2aily Transfers in Beef 
Infusion Broth Adjusted to j^ 7.0 Upon 
the R tmm o* E. »«^oi>athlae 



Infusion broth was prepared as in a prerlous 
perlaent, tubed in 10 ce* amount, and autoclared at 15 
pounds ppwisure for thirty minutes. The tubed oedla 
Incubated at 37* C. for 24 hoin-s for sterility. A nimber of 
the tubes irere inoculated wl13i six stabilized rough strains 
of Swine erysipelas, two stabilized rough strains of 
Salmonella suipestifer . and three stabilized rough straia« 
of Pasteurclla orgaolscis and Incubated at 37»C. They were 



d<J 





SI 

transferred in tubes of heef Infusion broth daily and at 
each transfer, a loopful from each culttire was stretfBB* on 
Bodified salts as«r plate« to determine the various type» of 

eolonies. 

fh» resiats are recorded on table 6. Dnrtag the fir«t 
fifteen days, all the cultures woTO still rou©b. EjoMaina- 
tion of colonies on modified salts a^BT plates at the twent- 
ieth transfer revealed a lalxture of R and I in all the j|g. 
rhusiopathiae cultures. The %^yMwfcii^ mtir>»«tifeT enltures 
were still rough, strains t and 8 of the Pasteurella or^in- 
IffMi »ere rough. Strain 17 at the twentieth transfer re- 
^•aled some colonies that may be either roxii^ (R) or inter- 
aa&lats U)* At «» thirtieth transplant, all the cultures 
of g. Tfmsiopethi&e revealed a mixture of S, I, and R foms 
of colonies. There were more emooth {s) colonies than rou^ 
(R) at this tlae. Strain 1146 ^re still rough. Strain 
1147 sho«a4 a mixture of R and 3 at the end of 30 daya« 
Strains 7, 8, and 17 of tlie Paatetirella orgeaiiams showed a 
mixture of R and I. All the 5^ rhusiqfs^fclma cultures re- 
farted to the S form at the fortieth transfer as observed 
«ft modified salts agar plates. All the strains of the 
SalWMiella and Pasteurella organ iaas ^cftmA a mixture of 
the different types of colonies with H still prevailing in 
number. 






Table 6« A oomjWLrison of the effect of beef 
Infusion broth (pIT 7.0),, and 10 per cent 
hraiolosous R issoEQiMi aeroR broth tit>on 
tto R form of E, rhuslopathlae. 



















Period of 


incubation (days) 


10 


20 


30 




40 


50 


Strain 


Media used 












106 




R 


R»I 


S,I.R 




S 


s 




TIWHiin •Brum 


H 


a»i 


S.I.R 




S 


s 


2310 


Infusion broth 


B 


E.I 


S,I,H 




S 


s 




Twmwi serum 


R 


R,I 


S.I.R 




S 


s 


^06 


iBftoSloa broth 


B 


H,I 


S.I.H 




S 


s 




Zanns serwi 


B 


R,I 


S.R 




s 


s 


xm» 


Ii^slon broth 


R 


R,I 


S,I,B 




s 


s 




laanne aerun 


H 


R,I 


S.I.R 




S.I 


8 


14159 


Infusion broth 


B 


R,I 


S.I.R 




s 


B 




TiTBfflmi •era 


S 


Rfl 


sli 




s 


ft 


PK 


Infusion broth 


B 


H,I 


S,R 




s 


8 




£MRBie sertaai 


R 


R,I 


S.I.R 




s 


S 


1146 


Infusion broth 


R 


R 


R 


Hi 


»I,S 


S.I 




Smune serum 


R 


B 


R 


R. 


,I,S 


S.I.R 


1147 


Itf^ttsion broth 


R 


B 


B.S 


H, 


iI.S 


R.I.S 




IGmmbs tt&wwt 


B 


B 


R,S 


B, 


,1,8 


R.i.s 


f 


tatwBitm toPBiat 


B 


B 


R.I 


R. 


,I,S 


S.I 




JwKttMt wHras 


B 


B 


R.I 


R. 


►i.s 


S.I 


• 


faftwim tito^t 


B 


S 


R,I 


R, 


,I,S 


s 






B 


B 


R.I 


H, 


J.s 


a 


17 


Infusion brotb 


• 


1 


R,I,S 


R« 


,1,3 


9 




ansas aeztiB 


R 


H.I 


8.1 


H, 


rl.S 


9 





All stralas of th« g, rhtialopathiae me?© still eeiootSi 
m% Hie fiftieth transfer. Strains 8 and 17 of the Pasteur- 
ella orsanisns were also fcrand to be 100 per cent fflaooth at 
this time. Strain 7 still retealed a fetr lnt«ra»diste (I) 
colonics with the S type preTalllng in nu-^ber. Strain 1146 
^ iWlwonella ggipestlfer still aSicMraS some Interraedla^ 
(I) colonies bat there WBSe wspd of the S t^^-pes. More R 
colonies were still found in 1147 at the end of 50 days. 

T!ie saooth (S) farms of E. Klaus iopathlae which were 
o^teftMHA ttCR the H Tariants •actauid to have all the char~ 
acterlBtlcs of the original S colony* 

The Effect of Daily Transplanta ill Ten Per Cent Ho«dCI<^ous 
1 Immmfi Ser^ra in Broth m^on tbe ft 

Form of E, 



One cubic centineter of hGaaolo^us R ixBOBine seruK 

Ly added to 9 cc» nutrient broth plttMA Hi a test 
tube, ilfter the media isere tested for atorility, they were 
inoculated with stabilized rough strains of E» rhugiottrtfciiM 
SalBonella sujpestifer and Pasteurella oi^aniema and inemf 
bated at 37" C, Dally transplants were made for a period of 

fifty days. Daily observation of thfe types of colonies on 
■i^if led salts agar plates icahlch were s treated with the 
cultures at every transplant mm ftade« 






Mm iftkom OB table 6, the 10 per cent honolosous R iah» 
Wtmm tminm in nxitrlGnt broth is just as effective a anfttm 
mm tlie beef Infttsion broth in produciag a E to S reversion 
of the Swin© ersrslpelas, 

*Pb6 Effeet of Daily Transplants in Semi-Solid 
Salts jigar Vpoa the B Tixm of 



Qra wssal-solid salts agar Tised in this investigation la 
a modification of the aedina described by Gcott in 1930, It 
naa prapaMd aiallar to the modified salts agar used through- 
out this study except that 0,1 per cent Bacto-agar i^ added 
to the alxture instead of 8 per cent. After the media ha»» 
been tnbed, aterilized and tested for sterility, they lere 
inoculated with stabilized rough strains of Swine erysipelas 
ddMSSildS. golpeatigBT «id fasEtaiirella or^aBisras« Transfers 
■sre aade dally for a period of 50 days. Observation of 
colonies on aodified salts agar similar to preeeeding ex- 
periments were also isede. 

Sa«l-solid salts agar was not effective in producing 
E to ii reversion. The B variants prevailed throushout the 



MiilBiifllBiaHMiHiiHil 



• 




TXllDL9RnE Of fMB 8 JIB) B W(XBMB OF 



to Iw « 9Bamg9X aeswmmt in tli« vovlai «f 
17 investlSKtom fhitt I3i» nng^ (B) f(am «r fui orgaalMi 
t« MPS virulent than the aMotli (S) tTpa* In 1916, Strikar 
qftnu'd tsiat tlai ^raviai of Tirulont pooonoooeei in lioaol- 
OfBMi iHBBO —111 l a roi w o o ft a teereaso in rimlcnoe, Orontt 
UiM) fowid his B type of hog ^kotmm IttotUno •• Ioini 
Tirolent then hio A typo* SoOlor in 19S7 otttod tbot tNi 
B tsrpo is fion Tintlo&t or at least loos Tirulent t!ian the 8 
fam* Ksloer U98S) c Osoi Pf od that the dlasooiatlon of an 
oHiiilsin t99m s to S wo of ton ooosopooloA igr a looo of 
▼irol«ioo« 

In l^K^ iblle Seals mam waipktas <» tho diooooiatioa of 
H f^Qont tHot llio A tmmm mm Tinaent for 
l^lgs while ^e R fonao orae hob vina^t. Li (19S9} 
SB tbo 4i«K>olation of ?t ffitliWftltfW* oboortoi thot 
the s foaa oso aero vimlont for lateratoyr aoioolo ISiob tkm 
1 foni. flosoiMi la IMS Ohile iroilEtoir on imsiiioiiiiiBiiiiB sta%» 
od tliat «» zever^Um froa fi to S alMitlter affaotod in tIto 
or in ritro aoo alooyo oo se apil a a %r aoqesiaition of maxi— 
■Bl irirtaonie. DtdoBOr (1MB) tonmA that the 3 foana of B^, 

SSSLklMKKKiAtL ^ ""^"^ ▼irulent than tho B fans* Bo akosro* 
od that the lethal doaao of B ooltws aas 15 tioss that of S 





^^IkH 



30 
etiltupe, Wilson ob»eir»»d In 1930 that the S forn of B. 
gejptaryojge mob aore iriiuLcnt than the H type. Ihlte (1S2S) 
dtated that smooth SalmoGella aiid irelated bacilli and fil- 
trates of brotli culture* were nore toxic to laboratory l«- 
4t&ts than were the rouf^ bacilli and filtrates, Button 
{19fB) fotBul that the S fsoxm of streptococci eas virulent 
and the R form of the organlflBS wt^e relatlrely avirulent. 
Vwagfwter in 192S obiNHrred that the E fom of B. anthracis 
«as awre virulent than the ^ form. Boaavor he atated that 
the B fonc was t2ie norsal type* 

This experiment 'esaa perforoed to deteiraine the relative 
viruleae* of th« 3 and B variants of S, ilni^|^^,te iao . 
Twrnty-four hour ctiltupea of six amooth (S)a&d six roa^ 
(R) strains of S. rhusior^thJaa. tao aaaootti and two roHg^ 
strains of ''nllSHlTi'' " £lii££Mi±!2£» ^^^ three smooth and 
ISttee rougli strains of Pasteurolla organiaos trere Injeeted 
In intrarausculerly into pl^gaens in various doses. 

The results of this exporlijant ware tabulated on tal0.a 
7. It was found as shomi on the table that ttie H fonM of 
Swine erysipelas were slightly aoro virulent for pigeons 
than the £• fcmu 'Phis obaervation is aonawhat in accord 
with the obaarwatlon of Himgester (1929) regarding %^ 
relative virulence of tlie B euad S types of B. anthraeis. 
table further abowB tbat the H f onie of Salaonella 





1^« A ooaapartson of the virulence of t*i« S, sbA 
R taroB ot E. rhusiOTsathlae by Inoculatli^ 
pigeons intramuscularly \9ith 24 hmir 
etilturGS of the variants. 



37 



ll»# <^ 




Dosase 








ptiMns 


C\iJ.titr© 


ec. 


Rorajlts 


75 


106S 


0.1 


Died 


in 


t d&ys 


76 




0.01 


Died 


in 


4 days 


77 




0.001 


Died 


in 


5 dfiye 


78 


106H 


0.1 


Died 


in 


3 dajrs 


79 




0.01 


Died 


in 


3 days 


80 




0.001 


Died 


in 


5 days 


81 


zsioa 


0.1 


Died 


in 


96 ho^rs 


82 




0.01 


Died 


in 


96 hoars 


83 




0.001 


Died 


in 


5 da^/s 


84 


2810B 


0.1 


Died 


in 


96 hours 


85 




0.01 


Died 


in 


96 hours 


86 




0.001 


Died 


in 


4 days 


87 


5S06S 


0.1 


Died 


in 


72 hours 


RR 




0.01 


Died 


in 


84 hours 


89 




0.001 


Died 


in 


96 hours 


90 


350da 


0.1 


Died 


in 


56 hours 


91 




0.01 


Died 


in 


72 hours 


92 




0.001 


Died 


in 


96 hours 


M 


JMBMBS 


0.1 


Died 


in 


65 hoiars 


«4 




0.01 


Died 


in 


65 hours 


95 




0.001 


Died 


in 


72 hours 


«l 


13843B 


0.1 


Died 


in 


St IMMBPS 


W 




0.01 


Died 


in 


60 otMlXHi 


98 




0.001 


Died 


in 


72 hotirs 


f9 


141593 


0.1 


Died 


m 


96 hours 


too 




0.01 


Died 


in 


96 hours 


101 




0.001 


Died 


in 


6 days 


102 


14159R 


0.1 


Died 


In 


72 hours 


103 




0.01 


Died 


in 


72 hours 


104 




0.001 


Died 


In 


4 days 


105 


pBS 


0.1 


Bled 


in 


96 hours 


106 




0.01 


Died 


in 


4 days 


107 




0.001 


Died 


m 


4 days 





(Table 7 contiiraed) 



108 


I)EH 


oa 


109 




0.01 


110 




0.001 


111 


U4tS 


0.3 


112 


1146B 


0.5 


113 


1147S 


0.3 


114 


1147H 


0.5 


115 


7S 


O.S 


116 


7H 


0.5 


117 


as 


0.3 


118 
119 


^ 


0»5 
O.S 


ICO 


17H 


0.5 



Died in 72 hours 

Died In 96 hotars 

Died In 9ti hours 

Oi«i in £4 hours 

Died in 36 hours 

Died In >j8 hours 

Died m S6 hours 

Lived 

Died In 48 hours 

Uvsd 





89 



stlfer and Pasteurolla organlsai wmm avlrulent «hile 
fSab S fonw wmre virulent tor pigeons, 

ms wnnsfft (m k mmTm m 'sm FittsBABiLiTT 

OF tHE S AND S fGBSB OF E. RHUSIOPATHIAE 
iXD ITS SffECT OH WS FILTIUfES 
IV wot TABIAHTS 



introAiotlon of Kendall (1232) of a aedlwi t^iet IM 
mm XiliWrtilj able to isolate froa InflaMis* orgs&iflwi biiiw 
lr.r n filter ^^sslng stw^re, also to deaonctrate filterable 
ana non fil-cerable for-„;s xn Imoteria led to his hypottidtfi* 
that all fcncwa Ijactcria can sind do exist in botli fllterabl* 
and non filterable states. 

Slnwi gfUHIinll Intiraated smacQisuxul results sitli hia 
woik. It was decided to determine the possible effect of K 
weSSmL on tlie f llterabll Ity of the S and R forsis of Swine 
eryslpelaa and its effect on tl^ filtrates of tbs imriants. 

K !g»aiTai -mm prepared as follows: Fresli ho(* intestines 
WKB expend, cleans and groimd In a asst chopper. The 
sroand raaterial was plft«sA in a jar oontalnlag ^oor toIi»ss 
of 95 ver swit ethyl alcohol and mm extracted at 37' C. for 
two days with oceaslooal stirring. The alcohol was replaced 
with fresh alcohol. This w«s isp ^ s tsd twice m^ing three 
«EtPBWstlons in all. The dry residue ^sas extracted with 





le. The betUBOBtf «a8 wmored by filtration and th» 
y ggi tt wi xmn erconeft out ob a glass plate, share a earrent 
of air G©J^e»at®^ by an electric fan playirti lapon the residue 
until it dried. The dried natericl was sround to a fine 
speeder in & mortar end «a8 Icept in a ^^11 stoppered contain^ 
er. 

The culture aediwi eee pmpared by adding S per cent by 
WBl^ght of the dried ground Intestine to modified Tyrode 
solution linvlns the following composition. 



HaCl •...••......•..••......• e*8 _ 

KCl ..« «•«•••••»« • 0,2 srtms 

QSCI ..••••• •••••• •••••««« 0»S fljTll 

lISC^lo** ••••••••••••••••••••••••••• ••• 0,1 flJfWHI 

lltt^^4 • ....•• 0.5 grasae 

WaSCOs .♦ ,, 0«S 

Glucose •••••••••••.••.•••• •••• 0.8 

Distilled raster , 1000.0 ce» 

A aaell anount of KaHCO- (0.5 gn, f^r every 1000 cc) was 
«iAed and the final reaction ras bro<ip;ht to pTT 7,4. The 
neditBB ees tebed In 10 and 50 cc» amounts and autoclaved for 
SO minutes at 15 pounds pressure. 

Before inoculation, ttie sterile It nedios ^es heeted in 
boilin"; TTater to driire out 12ie oxygen and then cooled rapid* 
ly in ice water, 

tm smooth and tiw rough strains of S. rhusio-o?.thiae 
one BBOoth and one rou^ strain of Salmonella suipestifer, 
«Hl one smooth and one ron^ strain of Pasteurella organism 
*ere Inoculated into sereral tubes of K media and incubated 





•t S7*C. for a period of one month. Snlxmltttres were made 
ttt «»#kly interrals and the cultures of tMii series were as- 
•Mftl«d and filtered throtigh BeiUcsf^ld If filter candles. 
The filtrates were inoculated Into several tubes of K media. 
They tjere incubated at 30*0. and at 37* C. sooe cultures were 
incubated aeroblcally and anaerobically at 37*C. The cul- 
tures were examined for signs of grovth at 24, 48, and 72 
hours, and at the end of one month before being finally dls- 
card€>d. aibcultures were made at freqi^nt intervals during 
period of observation. Control tubes of broth and agar 
wre inoculated from all filtrates. Utiinoculated 
of K asdlnft meem also included in efvefy series of 
culture tubes incubated to use as controls. 

BMftilts: Flltrable foma of Swine erysipelas, Sal-* 
taonella suipestlfer . and Pasteurella organisms were not pro- 
iBMi, There were no signs of growtli in tlra tobes of E 
medium inoculated with filtrates of the organisms. 

This experiment ccmfirtaed the woDcs of Craig and Jobxui 
(1932) and Carpenter and Long (1933), who attempted to 
dTxpll<»te t9*e work of Kendall. No Investigator has jret been 
able to duplicate the msrk of Zendall. Grinnell (1929) 
«%ilA»d that the reported filtration of such CBC^anisns as 
atR^toeocci bacilli of enteric gro\:^, dlph'Cherold bacilli^. 
and tubercle bacilli might be due rather to the pawaflt ot 

fmgiwnts of the bacteria In old cultures still capable of 





a 



m^TodjictXon or to a filtaration in a particularly favorable 
•OBpfHutlfle fluid than to the existence of a filtrable stige 
111 tte life cycle of the orgnnltwa 

A0OLUTIHATI(»r REACTIOHS 

Hit mnVL^mm «sea tn the ag^nt&ttation tests were pz«» 
ftred according to tfen^ nethod tKed by Schooling, Creech » 
ftttA Orey {1932}* Stabilized S asA S fontf of strains 1S845 
•ai SIM of Swine erysipelas organieas were need. Five- 
fwiths of a ctibic osntlss^er of a 24 hour nutrient broth 
eulturoBof selected variant* wm^ os e deA to eei^ mmfmraX 
COO ec« Erleniaeyer flasks containing sterile nutrient broth 
iB #00 cc. anoun^, adjusted to jE 7,2 and incubated at 37* €♦ 
for 48 hours. The broth cultures were eeatrif^igsd for one 
hour at xooderttii t^peed* the egpeznetent fliiSds dieeardsd 
miA the o^;BBtSMB inntiendcd in 0.4S per cent salt solution 
contalKllBS 0«S per cent p^eooL* forty-two inssEiredlBlts por 
cent saline instead of 0*85 per cent was used to 
iyeeteiteous clumping of the R v«rl«i««* theae aeie 
tor baaft mA centrifuged for cme hour* The sujjraanatHott 
fluids wmrn eeaifi €>««& off and the ovsttdana rest&Bpcnded 
in 0.42 per eent pheiKslized saline. «iis wsm regpeated 
three tii»«, after tfclch the antigens were stored in Urn 
ice box for future use. 





9h9 ^itiseara wn9 d««^op«A in rabbits by the injection 
«C «atig«iB prepared from four vterlafits of Swine ©rjwlpela*, 
tpo variants of J|iMH|y^ suites tifer and two variants of 
Pmt i u rella organiaias. The antigww wsxe prepared similar 
to 13kmm> wn»A for a^lutinatlon tests tsrithout tisins pheaolU 
fte imHIWMililMi wre 9A$a0%tA to tube 10 of IHV8rland*6 
iMi6«iic»eter« and heated at 60*C. for one hour, Tha btttta^ 
antigABe m^te tested for sterility by inoenXatins e^sr 
g|nntrf and lnc\&at«d at 37»C» for 48 hours. The initial 
dose TWJC 1 cc» injected subcutaneously into rabbits at 
•Bekly intervals wktiX five inoculaticais twre given, Tim 
^am «t0 doubled at eaeb musmmire inoculation. TwelvB 
mifB «ftar ISm last lit|»«tion, the rftbbits wrm bled fros 

oart, the blood allowftdl to clot, eentrlfueed, ant 
iMMRMtt WIS r^BGTtA vitb pipcttes anA fcq^ in the ice box 
tmtll vm.^ to be u»eA* 

All tests were incubated at 30*C. for 24 ho\ir» 
they 'sere read nacro aa a yt eelly* 

The xwRilts are recorded Jte table 8, The table 
that S, antlsera hf>v© higher titers than H antis^ra. 
antisera agglutinated 13843S swine erysipelas organisms in 
a dilution of 1:1280» asA 1181911 argenisaffi in a titer of 
itWO. 1S845B swine erysipelas WKf, aM^bt^lMtad llMfB 
in BW l lT i iw n iB a dilution of 1:640 and 15843S in a titrw of 
1:320. aCMS witiseza agglutinated S506S S«lne erysipelas 



43 





f 

i 

I 



•3 



I 

U 

o 



CO 

I 



». 



Pi 

o 

♦a 



4» 






CO 

s 

4» 

s 









o 



s| 



A 



I 

CO 



I § § 



o 



K 0; Pi ffi 

n « (O (0 

ffl 8 H 

n to H 



» 



CO 



000 

<o o <0 

H r-l H 



EO 03 

to 



2 CO 



cr; e 
to to 



a: 



H 



<0 



000 

ca CO fo 

ir> iH H H 



s 



•H »-4 «4 



£3 



H 



8 

to 



3 





05 

to 






p; 

00 






CO 05 
« CO 

H 
•-4 



05 M CD to 

3 I :S ® 

S 1 d 

H 





ill a titer of 1:640, and 35(>6R orsaniaas In ft 
titer of 1:60. S906R antia«« i^^utinated SdOftB orgmti— 
In a dilution of 1:520 and 5506S oT&Bnlmia in a titer of 
1:160. 1149 S ttstisara a^^iAlMt5d 1146S Sala<«iella sui^ 
rrfWTr'*W'*" is « dilution of 1:12^, and 1146B 
in a titer of 1:160. 1146B aatiftfffft ^gigplutinated 
iB a dilution of 1:640, and 1146S orgtfitaMI 
in a titer of 1:160. 8S antiseara agglutinflted 8S Paeteur- 
•11a organic* la a dilution of 1:640 and 8R orgaBioraa in a 
titOT of 1:160. 8R antisera agglutinated both 8H and 8S 
flqqpBtMS la a dilution of 1:160. 

Schutze (1921) stated that althoc^ little or no ag- 
glutination aay taica ptijiae aith a rou^^ strain of para- 
typhoid B, and its honologus snootTi seruia, the variant is 
not in i^i^trtirailDle, it trtll respond ttoII to a aeroa that 
ha-s been prepared trctt rou{?32 strain* Azionrig^t (1921) fotmd 
that B. ifaf^%m§am «sglutinat6d S organisms np to a titer 
of IjSSO and H oj^anisias to a titer of 1:80. Soule (1928) 
found that paratyphoid B S sera agglutinated 3 oj^pmisam 
in a titer of 1:2560 and R orgesniOEsa in a titer of 1:640. 
14. (1929) bbservod that the R fom of hog cholera baoillaa 
Imm a loaer titer than tha S type. Andreas (1922) stateA 
in hia aorls: on tha Sgl wiial la organ issffi that two antigens 
pcrasttit iriiich are not diatributed throughout the culture 



m 





«lft««d ttet t«e wU 4iftMi QtPM «>• fiaw t in etwty 

tVMght to ttm D ty^ r tawn t of Fatttal«gx «* Kanaaa State Col* 
lifi for dS4NPMMli»« AU tb«M 9i«lMHHi ««3P» WBom or leas 
of thB avttte typa of «&• AImsm* 9o t&r no ho^s havt 
iiwiialiffit ahull mf affaatat fUth mm mtmne tmrn at 
tfaa ilaaat aa iaaMrltiJ %r Oai«ar aii4l Oavtaa* 

flw loaloaa of tlM di a iai i aa«a aot aooatwit. In 
«o found itanonAaflaa (patattital \ m ti» kidneys. X& 
aaaaa «e fouad flwryaeltia aai yate a ti iotioa of tlia 
UMddBV INKI BO hMWinffiiijua fltt tl» KUaaya* Yba aoat aa»» 
•test laaftoDs f«mid vara Htm yaMa aa d pet^iaa ai tl» akia^ 
«r tfea ifioii glaada* aongaatioft and tUt^tLf 
MMMHOar dacanaration of ttm h aaa rt aad 
aUe^Uy thioliaaad iMaart valvaa* 

HtavaaaaylaiAlyt tarn hmaet valvaa aaaa HiSakaaadt 

•ww aw^^^ wnf^t^Htf awfc a^^BUBBwai^^waaUHpw^i^w •••^w ^^a^ni^^^iva ^^^t^^ aaaaaippa^aab^ ^^^p^^^Mfc^^Bia 

lalanatira aaasaatloa and fev acaaa of liaaaailMiNu va 

m asteaad^M davnal fls^ auibaat inaaiia aaaoaa^lon* Xa*' 





tensive areas of subcutaneous henoj 
•Uiht luiilllmtiw occurred in the dema. 

The above finding ipexe aoMiAMit in accord with the ob> 
serrations of ^a^lngton (1933) and Galger end Davlea (1932). 



A study warn aede of the Influence of eertaln liquid 
media in Inciting dissociation. After the variaRts of S. 
rhusjopath ia» i»Te obtali^d, studies were made on their 
parative wsiphology as well as their serological and physi- 
flfta^Ml properties. Studies trere also ismde on the in- 
fluence of certain liquid and semi -sol id media in inducing 
to smooth rercrslon. The f iltrablllty of the rough 
flMOOth strains of E. rhuslopathiae was also observed 
BS well as the influence of K medium on the filtrate. The 
pathology of the disease in swine 3?esultins frcaa B^ 
rhusiopathiae infection was studied laacroscopioally md 
roscoplcally. 

Six strains of smooth forms of Mffalpelothrix rhus lo- 
pe "Qiiae. two strains of Salmonella suipestifer . and t«» 
atralns of Pasteurella organiaaie were used in inoculating 
various liquid media. The SpJiaonella suipestifer and Pas- 
teurella organ iaos were used as a che(& or c<»itrol to see 
if the Swine erysijjelas organisms t^have similarly. 





n* different tfpmt of asloa^s produced were 
eft a» flBGOtSi (S)» intern^diato (I}» and ro«^ (B). 
Svine erysipeXafi orsanlsras were dissociated in l««a tiwi 
than were the S^^Iiaonella gglpestifsr azid Pastetorella opcan- 



Nutrlent broth, 5 per cent peptone and beef Infusion 
feroth, adjusted to pH 7*0, pH 7»6, ax^d pH 7,0 were all ef- 
fectlTG in inOucins 3 to B dissociation. Five per cent pep- 
tone isas acre effective than 1 pep cent peptone. T&a. per 
cent hfXBiolOfMMI tMMMl ««rum in broth also produced rou^li 

Several successive transfers of the rough (R) fozm in 
beef infusion broth adjusted to pH 7»0 oauset a K to S re» 
version. Daily transfer of the rou^ form In 10 pep eont 
hcmologous imsune mnimbk in broth ctLso ppoduead a H to S re- 
version. se(cii<-solid salts apof «&8 aot an effective meftiwi 
in inciting a R to S peve3?0i<m. 

tlwre «Bi8 BO indlfiNsttlon of bacteriophage in laany plat«« 
examined. 

The virul«w» of the au>&tii (S) end rou^ (B) types 
«MP» t»Bted and it nas found that they both kill pigeons in 
tlbcmt t2i« mmB ttee. It m^ fom&, 1 hu i wi>u r» that the H form 
«M sli^tly more virulent than the S ^rp»» tb& variants 
of S. liiusiopathiae do not behave like the variant© of Sal- 





HffBolla sulpestlfer and Pasteurella organ isaffi in this re- 
spect. The roucb (R) foims of Salacmella sulpestifer and 
^«i1»t)trella orsanlsias are less virulent than the saaooth (S) 
typos. 

It was found that the roiigh (R) form of E» rhnsloiJathiae 
has a lover agglutisatioa titer than the smooth (S) form. 
It «auB also found that the saaooth (S) and rough (R) typee 
mtmtt a^lutiaate at losrer dilutions. 

The filtrabllity of the smooth (S) and rough (R) foiaaa 
wore teated on K laeditBH and tl»y were not found to be f 11-' 
trable« 

Qmi lesions found in Swine erysipelas were not constant. 
In 8000 «WM» wm tomtA heEJoriiiases (petechial) on the kid- 
neys. In other eases ^ found jpiuRryiiSitis and peteohiation 
of the bladder but no hmmaaPitmglBB on XSub kidneys. The most 
constant lesions found WKem the eoagestion of the lyaj^ 
glands, congestion of the spleen, slightly thickened heart 
valves and reddened patches on the skin. Microscopically 
wt fVHUid be9U>ri4iag«8 of the thloicened heart Vfilvea, Iieraor- 
rhascc under the skin, and consestion of the spleen. 





m 



0mcwstom 

Xm S» I0 and R ▼arlants of SwUm erjraipelas aay b© ob- 
tained by proper treafeaent, 

2, E. r!mslOT»athlae dissociated in less ttae than 
^Iiaonella suipestifcr and Pasteurella organiBEis* 

8. Laarge voltaae of liq[aid ctilture aedliim is isore ef- 
fect l-ro m Incitinnt S to H dissociation, 

4» i^iQg culture in nutrient broth of neutral or 
«2Jsaline reaction produced 3 to H dissociation, 

5, Five per cent peptone pro*iced a Greater ntssber of 
Tariante than one per cent pq^ione, 

6, Ten per cent iicaiiologous iiamtine MVttm in broth pro- 
duoad S to H dis0ocietion« 

7, Tfce production of E In different media fwa "tarinblei, 
8» H to S reversion was a««oBg)Ii«hed by daily transfer 

la beef infusion broth and 10 per eent homologous B imasBm 
seriaa in broth* 

©• S^ttl-a5lid agar 1b not as effective as beef in- 
fusion broth and homologous iwaine eerum broth in producing 
B to S reversion. 

10, The S foim derived from th© R variant h^d practi- 
cally all the characteristic of the original S type. 





11* Ito • «B^8oii warn ag^utlnatod by the 3 scrum In « 
btglte? titer than B antigen were asBlutlnated by the R sera. 

l£m S and B torma cross agglutinated at lover titer. 

13, Kie R fom of E. rhusio-paHit— wus sli^iitly mora 
Tirttient than the 3 fom, 

14« K. stadia had no effect in producing f iltrable stage 
In the B and S f ozimi* 

15. The individiial oella of the B nulture afpeaved to 
l»e loiter and slightly larger than the S fonite The cells of 
Hhi B irarianta appeared either in chains or in clximps vhll» 
Wmm of tbe S fom i^pfMtred singly* 

16* thm B colonies are larger and more irregular than 
t&e S colon lea. 

17. The pxMWMM of bacteriopha^B «afi not noted in this 
investigation* 

18, The pathology of the disease produced in swine as 
a result of 3S, iJms J'lKiti^e infection was not constant* 






52 



j|0S!fO1ILEI)C8Q9fT 

2 lAA to w p if*— aar sreat Indebtvdbiftse to Dr. B« 7* 
IdfiiilHirdty Read of the Dopsrtiaeiit of Pathology et Kaneae 
8t8t» Collie of Agriculture and Applied Selenee, Dr. L, D, 
Bushnell, Heed of the Departiaent of Bacteriology, asA 
Drs. J. P, Scott and E. E, Leasure, both of the Departaent 
of Patholocsy, iflio by tlielr onerous adriee, help and sug- 
(|»Btlons helped aa greatly In the prosecution of this in- 
vestigation. 





ufSMfUHB cxm 



stiAliM OB wttmp agglutiiiatlon !• Tbm ililwMUllii 
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INffttttloa in bMtwia la Mlatioo to ag^attaaticm 
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MsikmnAtt J« A«, «|A Pitt» R. m 

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J»9 find Ciuoa. ?l« Cited ^ I!«dli«7» P« 
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ABtM9*4M« 1MB« 

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FBtltim to ffpotiiB flltr&hle tomm of 
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U»d«» a9ldtJb«Att. 19»U 



MthocpiiB in SBBdBll*s oodia* iToo. soe* 
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rtlbUity of "B* and "S"* Ugm of 
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Vhi fcntorliirtiiipi bbI it* ^albHwim* TmiOBtoA lor 
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Hi 



53 





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r • 




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